Flow Cytometry Core
Stephen Perfetto, M.S., MT(ASCP)
Chief, Flow Cytometry Core
Major Areas of Research
- Development, advancement, and application of novel multidimensional and multicolor flow cytometry techniques
- Detailed assays to an in vivo system to explore the role of CTL in lymphocyte dynamics during viral replication
- Identification of viral reservoirs in CD4 T-cell subsets
- Sequence analysis of viruses isolated from specific CD4 T-cell subsets give us an understanding of the spread of virus through the CD4 compartment and the contribution of different CD4 subsets to both active viral production and latent reservoirs.
- Analysis and sorting of HIV antigen-specific B cells to study neutralizing antibodies and to correlate the immunophenotyping of these cells using index sorting
The Vaccine Research Center (VRC) Flow Cytometry Core Facility (FCC) works closely with all of the immunology laboratories at the VRC, managing a range of instruments from advanced analyzers to the most sophisticated cell sorters. The FCC serves the research community as well as the clinical trials supported by the VRC. This facility has become an internationally recognized entity, a world leader in the development and application of multidimensional and multicolor flow cytometry. The FCC has collaborated on or directly developed the necessary technologies to perform 20-parameter flow cytometry, analysis, and sorting. This technological feat required the combined development of novel antibody-conjugated fluorochromes, including nano-crystal development hardware (the integration of multiple lasers and detectors, with the associated high-precision optics), software tools (for complex multidimensional data analysis), and validation techniques (for instrumentation and reagents quality control).
The FCC currently manages seven instruments with capabilities of serving a wide range of clinical and research demands: 1) five LSR II instruments equipped with high-throughput robotic units and capable of measuring 20 parameters in a fast-acquisition mode from specimens loaded on a 96-well tray; 2) two FACSAria II instruments, each capable of 20-parameter cell measurements at high-speed sorting (20,000 to 60,000 cells per second), one located in a biosafety level (BSL)-2 environment for non-infectious cell sorting and one located in the BSL-2 plus, capable of infectious cell sorting. The FCC facility leads the development and advancement of cytometric technology at all levels: instrumentation, reagents, detection, and analysis.
For more information on research conducted by Mr. Perfetto, visit the ImmunoTechnology Section.
Stephen P. Perfetto received a B.S. degree in medical technology from West Virginia University in 1977 and completed his M.S. degree in 1981 from West Virginia University. He studied and worked in the clinical blood bank and clinical immunology laboratories until 1988, when he joined the EPICS Division of Coulter Corporation. In 1990, he was recruited to the Walter Reed Army Institute of Research (WRAIR) and was the manager of the core flow cytometry facility. While at WRAIR, he was involved in large HIV vaccine trials and developed functional tools to study the immune system of infected individuals.
In 2000, he joined the VRC as a staff scientist and manager of the flow core facility. This facility is the world leader in multicolor flow cytometry and continues to develop this technology actively on a number of different fronts; one focus is on hardware development and reagent and analysis development. For several years, this lab has collaborated to develop Quantum Dots for use in immunophenotyping experiments. The advent of these fluorochromes provided an enormous advance in multicolor technology, allowing us to proceed from 12-color to 18-color very quickly. This group is actively working on new data analysis techniques: One major focus is the analysis and presentation of metadata (for example, summarizing our functional analysis in which they have broken down each single response into hundreds of categories defined by the expression patterns of individual cytokines or other functional measurements). Another focus is on particular aspects of immune function or viral dynamics, within the context of the major research efforts.
Perfetto SP, Ambrozak DR, Nguyen R, Roederer M, Koup RA, Holmes KL. Standard practice for cell sorting in a BSL-3 facility.Methods Mol Biol. 2011;699:449-69.
Wu X, Zhou T, Zhu J, Zhang B, Georgiev I, Wang C, Chen X, Longo NS, Louder M, McKee K, O'Dell S, Perfetto S, Schmidt SD, Shi W, Wu L, Yang Y, Yang ZY, Yang Z, Zhang Z, Bonsignori M, Crump JA, Kapiga SH, Sam NE, Haynes BF, Simek M, Burton DR, Koff WC, Doria-Rose NA, Connors M; NISC Comparative Sequencing Program, Mullikin JC, Nabel GJ, Roederer M, Shapiro L, Kwong PD, Mascola JR. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science. 2011 Sep 16;333(6049):1593-602.
Zarkowsky D, Lamoreaux L, Chattopadhyay P, Koup RA, Perfetto SP, Roederer M. Heavy metal contaminants can eliminate quantum dot fluorescence. Cytometry A. 2011 Jan;79(1):84-9.
Chattopadhyay PK, Perfetto SP, Yu J, Roederer M. The use of quantum dot nanocrystals in multicolor flow cytometry. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010 Jul-Aug;2(4):334-48.
Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M. Amine-reactive dyes for dead cell discrimination in fixed samples. Curr Protoc Cytom. 2010 Jul;Chapter 9:Unit 9.34.
Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Quality assurance for polychromatic flow cytometry. Nat Protoc. 2006;1(3):1522-30.